A major goal of microorganism detection research is to develop inexpensive, fast, reliable, and sensitive detectors. Standard laboratory procedures are currently available for the detection of microorganisms. The vast majority of procedures are based on the use of agar media on which specific microorganisms grow over a period of time. The normal incubation period is between 24 to 48 hours. After the microorganisms multiply their presence can be identified and quantified.
The main drawback of the existing tests is the time required for obtaining their results. Bacterial contamination in water sources results in the shutdown of water sources and systems and requires the use of more expensive water supply alternatives. Fast detection of microorganisms is needed to allow for shorter shutdown periods. In the medical sector, bacterial identification and antibiotic sensitivity tests are required in any medical situation in which antibiotics are to be administrated. The time required for obtaining test results is between 72 to 96 hours. Reducing this time period will produce better results and faster patient recovery. In the food and beverage industry, raw materials and manufactured goods are routinely inspected for bacterial contamination. The required incubation period for test results does not allow for immediate process treatment and causes delays both in the manufacturing and supplying of goods. Reducing the taking time period can result in savings in infrastructure and labor.
In the last few years several methods were developed in order to identify and enumerate bacteria in 1.5 to 11.0 hours. Methods that are relatively simple require a large amount of bacteria (above 104 in 1 ml sample), while methods that can detect small numbers of bacteria are expensive and cannot be used as large scale systems.
U.S. Pat. No. 5,811,251 discloses a system for counting the number of viable microorganisms based on a CCD system. However this system cannot differentiate between different types of bacteria and provides only a total number of bacteria. U.S. Pat. Nos. 5,972,641 and 5,518,894 disclose a rapid coliform detection systems using a statistic methods for determining the number of bacteria. Said methods require up to 11 hours for obtaining the results in low number of bacteria. Other patents disclose a method for detection of microorganisms using fluorescence and laser light source (U.S. Pat. Nos. 5,891,394, 5,858,697, 5,763,203, 5,751,839 and 5,663,057). The disadvantages of said methods is the use of an expensive laser light source and the detection of microorganisms directly from the filter which is not smooth and causes problems during analysis. In addition, these systems are not portable and are relatively expensive.
Immunoassay methods are also used for detecting certain types of microorganisms (Lee et al., App. Environ. Microbiol., Vol. 56, pp.1541-1546). In these methods, specific antibodies labeled with a fluorescent or radioactive dye are used to detect the microorganism. However, immunoassay methods are limited in that they require the production of antibodies against each microorganism of interest, which is time-consuming and expensive.
“Sol-gel” is the term used to indicate inorganic glass manufactured at room temperatures based on metal oxides. A certain process involving ceramic materials in which the sol (solution) is transformed to a gel phase through hydrolysis, condensation and polymerization. The common starting materials for the sol-gel preparation are ormosils or metal oxides. In recent years sol-gel has been applied to organosilanes to create “glass at room temperature”. Sol-gel type materials comprise pores ranging from tens of angstroms to tens of nanometers, and exhibit a large area to mass ratios e.g., hundreds of square meters per gram. Sol-gel materials are transparent even at UV wavelengths, and are simple to prepare in different shapes, such as powders, monolithic blocks, thin sheets, fibers etc.
The use of sol-gel-based materials to entrap various organic molecules in a matrix media was described in the art. (Avnir et al., Supramolecular architecture in two and three dimensions Bein T. (ed.) American Chemical Society Symposium Series XXX, 1992). Using said technology, organic molecules are entrapped at room temperatures within the sol-gel matrix without impairing the structure of the relatively sensitive organic molecule. In addition, the entrapped molecule retains almost all of the original physical and chemical characteristics, and is available to outside reactants as a result of the massive pore system inside the sol-gel.
U.S. Pat. No. 6,022,748 discloses a method for the direct detection of analytes using color changes in response to selective binding of analytes to a surface. Said detection occurs in immobilized biopolymeric material encapsulated into metal oxide glass using the sol-gel method. The disadvantages of this method are that only large amounts of bacteria can be detected or enumerated, since only high counts are able to cause a visible color change in the sol-gel. Furthermore, said method cannot differentiate between viable and non-viable microorganisms, since it is based on the binding of the microorganisms to the sol-gel surface, independent whether said microorganisms are viable or not.
Armon et al. (J. of Biotechnology 51, 279-285) disclose a method for detecting large quantities of E. coli bacteria by spreading them on sol-gel doped with specific compounds, said compounds being uptaken into the bacteria, which consequently causes the bacteria to glow at specific wavelengths. However, this method does not provide a method suitable to count bacteria present in a given sample. In addition, detection of low number of microorganisms is not possible using said method.
The art has so far failed to provide a fast method for the enumeration of microorganisms, which is sensitive enough to provide a reliable count at low microorganism concentrations.
It is a purpose of this invention to provide a fast and sensitive method for the detection of viable microorganisms.
It is another object of the invention to provide a method and compositions useful in providing an enumeration of microorganisms found in low-count samples.
Other objects and advantages of the invention will become apparent as the description proceeds.